Therapeutic use of a number of peptides, polypeptides and proteins is curtailed because of their immunogenicity in mammals, especially humans. For example, when murine antibodies are administered to patients who are not immunosuppressed, a majority of such patients exhibit an immune reaction to the introduced foreign material by making human anti-murine antibodies (HAMA) (e.g. Schroff, R. W. et al (1985) Cancer Res. 45: 879-885; Shawler, D. L. et al (1985) J. Immunol. 135: 1530-1535). There are two serious consequences. First, the patient's anti-murine antibody may bind and clear the therapeutic antibody or immunoconjugate before it has a chance to bind, for example to a tumor, and perform its therapeutic function. Second, the patient may develop an allergic sensitivity to the murine antibody and be at risk of anaphylactic shock upon any future exposure to murine immunoglobulin.
Several techniques have been employed to address the HAMA problem and thus enable the use in humans of therapeutic monoclonal antibodies (see, for example, WO-A-8909622, EP-A-0239400, EP-A-0438310, WO-A-9109967). These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanized” antibodies have, in several cases, still elicited an immune response in patients (Issacs J. D. (1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999) Transplantation 68: 1417-1420).
A common aspect of these methodologies has been the introduction into the therapeutic antibody, usually of rodent origin, of amino acid residues, even significant tracts of amino acid residue sequences, identical to those present in human antibody proteins. For antibodies, this process is possible owing to the relatively high degree of structural (and functional) conservatism among antibody molecules of different species. For potentially therapeutic peptides, polypeptides and proteins, however, where no structural homologue may exist in the host species (e.g., human) for the therapeutic protein, such processes are not applicable. Furthermore, these methods have assumed that the general introduction of a human amino acid residue sequence will render the re-modeled antibody non-immunogenic. It is known, however, that certain short peptide sequences (“T-cell epitopes”) can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatability complex (MHC) in order to trigger the activation of T-cells. For peptides presented by MHC Class II, such activation of T-cells can then give rise to an antibody response by direct stimulation of B-cells to produce such antibodies. Accordingly, it would be desirable to eliminate potential T-cell epitopes from a peptide, polypeptide or a protein. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include therapeutic use of granulocyte-macrophage colony stimulating factor (Wadhwa, M. et al (1999) Clin. Cancer Res. 5: 1353-1361) and interferon alpha 2 (Russo, D. et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413).
The elimination of T-cell epitopes from proteins has been previously disclosed (see, for example, WO 98/52976, WO 00/34317). The general methods disclosed in the prior art comprise the following steps:
(a) Determining the amino acid sequence of the polypeptide or part thereof
(b) Identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by any method including determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays.
(c) Designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in turn, modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope.
(d) Constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties.
Other techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides and able to bind to T-cell clones from peripheral blood samples from human or experimental animal subjects have been used in the art [Kern, F. et al (1998) Nature Medicine 4:975-978; Kwok, W. W. et al (2001) TRENDS in Immunology 22: 583-588] and may also be exploited in an epitope identification strategy.
The potential T-cell epitopes are generally defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Such potential T-cell epitopes can be measured to establish MHC binding. Implicit in the term “T-cell epitope” is an epitope which when bound to MHC molecules can be recognized by the T-cell receptor, and which can, at least in principle, cause the activation of these T-cells. It is, however, usually understood that certain peptides which are found to bind to MHC Class II molecules may be retained in a protein sequence because such peptides are tolerated by the immune within the organism into which the final protein is administered.
The invention is conceived to overcome the practical reality that soluble proteins introduced into an autologous host with therapeutic intent, can trigger an immune response resulting in development of host antibodies that bind to the soluble protein. One example amongst others is interferon alpha 2 to which a proportion of human patients make antibodies despite the fact that this protein is produced endogenously [Russo, D. et al (1996) Brit. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413]
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins and the major focus of the present invention. However, isotypes HLA-DQ and HLA-DP perform similar functions, hence the present invention is equally applicable to these. MHC HLA-DR molecules are homo-dimers where each “half” is a hetero-dimer consisting of α and β chains. Each hetero-dimer possesses a ligand binding domain which binds to peptides varying between 9 and 20 amino acids in length, although the binding groove can accommodate a maximum of 9-11 amino acids. The ligand binding domain is comprised of amino acids 1 to 85 of the α chain, and amino acids 1 to 94 of the β chain. DQ molecules have recently been shown to have an homologous structure and the DP family proteins are also expected to be very similar. In humans approximately 70 different allotypes of the DR isotype are known, for DQ there are 30 different allotypes and for DP 47 different allotypes are known. Each individual bears two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and such structures point to an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide [Brown et al Nature (1993) 364: 33; Stern et al (1994) Nature 368: 215]. Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
There is a considerable amount of polymorphism within the ligand binding domain with distinct “families” within different geographical populations and ethnic groups. This polymorphism affects the binding characteristics of the peptide binding domain, thus different “families” of DR molecules will have specificities for peptides with different sequence properties, although there may be some overlap. This specificity determines recognition of Th-cell epitopes (Class II T-cell response) which are ultimately responsible for driving the antibody response to B-cell epitopes present on the same protein from which the Th-cell epitope is derived. Thus, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition which is a function of the peptide binding specificity of that individual's HLA-DR allotype. Therefore, in order to identify T-cell epitopes within a protein or peptide in the context of a global population, it is desirable to consider the binding properties of as diverse a set of HLA-DR allotypes as possible, thus covering as high a percentage of the world population as possible.
A principal factor in the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cell via presentation on MHC class II molecules. In order to eliminate or reduce immunogenicity, it is thus desirable to identify and remove T-cell epitopes from the protein.
The unmodified biological molecules can be produced by recombinant technologies, which are per se well known in the art, using a number of different host cell types.
However, there is a continued need for analogues of said biological molecules with enhanced properties. Desired enhancements include alternative schemes and modalities for the expression and purification of the said therapeutic, but also and especially, improvements in the biological properties of the protein. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide the selected biological molecule with reduced or absent potential to induce an immune response in the human subject. Such proteins would expect to display an increased circulation time within the human subject and would be of particular benefit in chronic or recurring disease settings such as is the case for a number of indications for said biological molecule.